1. Field of the Invention
The present invention relates to a probe set for detecting a target substance in a sample and a method of detecting a target substance by using the same.
2. Description of the Related Art
Conventionally, Southern hybridization, Northern hybridization, in situ hybridization, etc. have been well known as a method of detecting a target nucleic acid in a sample. These are methods of detecting the presence and/or position of a target nucleic acid by hybridizing a labeled probe with the target nucleic acid which is blotted in a membrane or is in a fixed cell, and then detecting the signal generating from the labeled probe. These methods do not involve amplification of a target nucleic acid, so that particularly when the amount of the target nucleic acid in a sample is very low, the signal is weak and the detection sensitivity is low.
As one method of detecting a nucleic acid in a sample by amplifying the nucleic acid, a method described in U.S. Pat. No. 5,770,408 can be used. This method involves the following steps. A target nucleic acid-containing sample, probe A, and probe B are mixed to prepare a reaction mixture. Probe B is hybridized cyclicly with the target nucleic acid, and the 5′- and 3′-ends of probe B are ligated by a ligase with each other. The reaction mixture is heated to a high temperature of 90° C. or more to thermally denature the hybridization product of the target nucleic acid and probe B. Upon thermal denaturation, the hybridization product can have a structure wherein the single-stranded target nucleic acid penetrates the cyclic probe B. By decreasing the temperature of the reaction mixture, the probe A is hybridized cyclicly with the cyclic probe B, and the 5′- and 3′-ends of probe A are ligated by a ligase with each other. In this manner, the probes A and B are bound linearly to one molecule of the target nucleic acid. The probes A and B bound to the target nucleic acid are labeled, whereby the nucleic acid contained even in a very small amount in a sample can be detected with a strong signal.
However, the target in the above method is limited to nucleic acid, and other substances such as protein cannot be detected. In addition, the step of thermally denaturing the reaction mixture at a high temperature of 90° C. or more is necessary, thus necessitating an apparatus for keeping the reaction mixture at a high temperature for a predetermined time.